Reduced heart rate variability is associated with altered clinical laboratory profile in people living with HIV.

Background: We compared heart rate variability (HRV) indices between people living with HIV (PLWH) and HIV-negative individuals to ascertain the independent association between HIV infection and reduced HRV, and further investigated whether distinct clinical laboratory profiles exist between PLWH with and without reduced HRV. Methods: This cross-sectional analysis included 304 PLWH and 147 HIV-negative individuals with comparable age and sex. Thirty-two routine clinical laboratory indices (including hematology and biochemistry) closest to the survey were extracted from the Electronic Medical Record System. HRV indices were divided into two categories: low (lowest quartile, Q1) and moderate-to-high (combined, Q2‒Q4). Results: The time domain indices, ln(SDNN), ln(RMSSD), and ln(PNN50), as well as the frequency domain indices, ln(HF), ln(LF), and ln(VLF), were all significantly reduced in PLWH versus HIV-negative individuals (all p < 0.05). These associations remained for ln(SDNN), ln(PNN50), ln(HF) and ln(LF) even after adjusting for potential confounders in multivariable models. PLWH with low HRV indices exhibited distinct clinical laboratory profiles that were characterized by an elevation in fasting plasma glucose, white blood cell count, neutrophil count, neutrophil%, and a reduction in albumin, total protein, urine creatinine, lymphocyte%, red blood cell count (RBC) and nadir CD4 count. The final stepwise logistic regression models for low SDNN included older age, decreased total cholesterol levels, elevated neutrophil count, and the use of antidiabetic medications, whereas the final model for low LF included older age, reduced RBC and the use of antidiabetic medications. Conclusion: PLWH exhibit impaired parasympathetic activity, as evidenced by reduced SDNN, PNN50, LF and HF. Furthermore, PLWH who have reduced HRV indices exhibits distinct clinical laboratory profiles that are related to systematic inflammatory response and diabetes.

Clinical aPDT's effect on Candida albicans: Antifungal susceptibility, virulence gene expression, and correlation with leukocyte and neutrophil counts.

BACKGROUND: Our previous clinical trial demonstrated that antimicrobial photodynamic therapy (aPDT) with methylene blue (MB) and potassium iodide (KI) effectively killed Candida albicans (C. albicans) in adult AIDS patients with oral candidiasis, regardless of biofilm formation or 25S rDNA genotype. This study evaluated changes in antifungal susceptibility and virulence gene expression in C. albicans before and after aPDT, and explored factors related to clinical aPDT efficacy. METHODS: Twenty-one adult AIDS patients with C. albicans oral candidiasis were divided into Group a (400 µM MB, N = 11) and Group b (600 µM MB, N = 10). Both groups received two aPDT treatments, where MB was applied for 5 min, followed by 300 mM KI, and illuminated for 30 min (37.29 J/cm²). C. albicans isolates were collected before and after treatment to assess antifungal susceptibility (fluconazole, itraconazole, flucytosine, amphotericin B) and gene expression (CAT1, HWP1). Peripheral blood tests were analyzed for correlations with aPDT efficacy. RESULTS: aPDT reduced minimum inhibitory concentration (MIC) values for amphotericin B, fluconazole, and flucytosine, with significant reductions primarily after the first treatment. MIC reductions differed between groups, with Group a showing greater decreases in flucytosine and fluconazole MICs, and Group b in amphotericin B MICs. No significant changes in CAT1 or HWP1 expression were observed. Clinical efficacy of aPDT negatively correlated with leukocyte and neutrophil levels. CONCLUSIONS: aPDT effectively reduces MICs of antifungal drugs against C. albicans isolated from treated patients, particularly after the first treatment. The concentration of MB required to reduce MICs varies among different antifungal drugs. aPDT does not alter CAT1 or HWP1 expression, and its clinical efficacy in eradicating C. albicans is negatively associated with leukocyte and neutrophil levels.

Key role of activated platelets in the enhanced adhesion of circulating leucocyte-platelet aggregates to the dysfunctional endothelium in early-stage COPD.

Background: Chronic obstructive pulmonary disease (COPD), usually caused by long-term tobacco smoking, is independently associated with systemic inflammation. However, little is known about the systemic inflammatory status of patients with early-stage COPD (classified as GOLD 1) and long-term smokers with normal lung function (LF). Here, we characterised the early changes in the associated inflammatory state in patients with GOLD 1 and in long-term smokers with normal LF. Methods: Fresh blood samples from 27 patients with GOLD 1, 27 long-term smokers and 14 non-smokers were analysed. Results: Ex vivo blood analysis revealed greater leucocyte-platelet adhesion to TNFα-stimulated pulmonary endothelium in patients with GOLD 1 than in smokers and non-smokers. In addition, platelet reactivity (platelet count and activation, and fibrinogen levels) and the frequency of leucocyte-platelet aggregates were higher in the GOLD 1 group than in the other groups. Some of these findings correlated with the severity of lung dysfunction, while platelet hyperactivity correlated positively with leucocyte-platelet adhesion. The GOLD 1 group also had a higher Th17/Treg ratio and higher circulating levels of IL-17C and C-reactive protein than the other groups. However, long-term smokers also had higher leucocyte counts and activation, and higher plasma levels of TNFα and IL-6 than non-smokers. Conclusion: Our data suggest that the altered inflammatory parameters in long-term smokers may represent early biomarkers of COPD. Accordingly, peripheral immune monitoring based on the above parameters may be useful to prevent disease progression in long-term smokers with normal LF and early COPD.

Effects of perfluoroalkyl sulfonic acids on developmental, physiological, and immunological measures in northern leopard frog tadpoles.

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 µg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 µg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 µg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 µg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg-1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds.

PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus).

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

A Flow Cytometry-Based Examination of the Mouse White Blood Cell Differential in the Context of Age and Sex.

Analysis of the white blood cell differential as part of a flow cytometry-based approach is a common routine diagnostic tool used in clinics and research. For human blood, the methodological approach, suitable markers, and gating strategies are well-established. However, there is a lack of information regarding the mouse blood count. In this article, we deliver a fast and easy protocol for reprocessing mouse blood for the purpose of flow cytometric analysis, as well as suitable markers and gating strategies. We also present two possible applications: for the analysis of the whole blood count, with blood from a cardiac puncture, and for the analysis of a certain leukocyte subset at multiple time points in the framework of a mouse experiment, using blood from the facial vein. Additionally, we provide orientation values by applying the method to 3-month-old and 24-month-old male and female C57BL/6J mice. Our analyses demonstrate differences in the leukocyte fractions depending on age and sex. We discuss the influencing factors and limitations that can affect the results and that, therefore, need to be considered when applying this method. The present study fills the gap in the knowledge related to the rare information on flow cytometric analysis of mouse blood and, thus, lays the foundation for further investigations in this area.

Ex Vivo Analysis of the Association of GFP-Expressing L. aethiopica and L. mexicana with Human Peripheral Blood-Derived (PBD) Leukocytes over 24 Hours.

Leishmania parasites are transmitted to mammalian hosts through the bite of sandflies. These parasites can infect phagocytic cells (macrophages, dendritic cells, and neutrophils) and non-phagocytic cells (B cells and fibroblasts). In mice models, the disease development or resolution is linked to T cell responses involving inflammatory cytokines and the activation of macrophages with the M1/M2 phenotype. However, this mechanism does not apply to human infection where a more complex immunological response occurs. The understanding of interactions between immune cells during Leishmania infection in humans is still limited, as current infection models focus on individual cell types or late infection using controlled human infection models (CHIMs). This study investigated the early parasite infection in freshly isolated peripheral blood-derived (PBD) leukocytes over 24 h. Flow cytometer analysis is used in immunophenotyping to identify different subpopulations. The study found that among the L. aethiopicaGFP-associated leukocytes, most cells were neutrophils (55.87% ± 0.09 at 4 h) and monocytes (23.50% ± 0.05% at 24 h). B cells were 12.43% ± 0.10% at 24 h. Additionally, 10-20% of GFP+ leukocytes did not belong to the aforementioned cell types, and further investigation revealed their identity as CD4+ T cells. Data not only confirm previous findings of Leishmania infection with PBD leukocytes and association with B cells but also suggest that CD4+ T cells might influence the early-stage of infection.

Development and evaluation of a radiolabelling agent for white blood cell scans.

Radiolabelled autologous leukocytes have been used for the clinical diagnosis of inflammation and infection. To develop a stable and efficient radiopharmaceutical for labelling leukocytes, we prepared a novel radioiodinated cell-penetrating peptide, 125I-TAT, using a bi-functional linker. 125I-TAT was stable for two days under three different temperature conditions of -20 °C, 4 °C, and 40 °C, with its radiochemical purity remaining over 99%. Iodinated TAT was non-toxic to leukocytes with an IC50 value of over 100 µM. The labelling efficiency of 125I-TAT using 1ｘ107 cells ranged from 27% to 53% when the three leukocyte cell lines were pre-treated with DMSO. This is comparable to the labelling efficiency recommended by the guideline for conventional labelling agents using 2ｘ108 cells. Radioiodinated cell-penetrating peptide may be an improved radiopharmaceutical for white blood cell scans by further optimization.

Comprehensive identification, isolation, and culture of human breast cell types.

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing, and a thorough dissection of the genes and pathways defining each cell type. While the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the 12 major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind-and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

Enhancers regulate genes linked to severe and mild childhood asthma.

Background: Children with severe asthma suffer from recurrent symptoms and impaired quality of life despite advanced treatment. Underlying causes of severe asthma are not completely understood, although genetic mechanisms are known to be important. Objective: The aim of this study was to identify gene regulatory enhancers in leukocytes, to describe the role of these enhancers in regulating genes related to severe and mild asthma in children, and to identify known asthma-related SNPs situated in proximity to enhancers. Methods: Gene enhancers were identified and expression of enhancers and genes were measured by Cap Analysis Gene Expression (CAGE) data from peripheral blood leukocytes from children with severe asthma (n = 13), mild asthma (n = 15), and age-matched controls (n = 9). Results: From a comprehensive set of 8,289 identified enhancers, we further defined a robust sub-set of the high-confidence and most highly expressed 4,738 enhancers. Known single nucleotide polymorphisms, SNPs, related to asthma coincided with enhancers in general as well as with specific enhancer-gene interactions. Blocks of enhancer clusters were associated with genes including TGF-beta, PPAR and IL-11 signaling as well as genes related to vitamin A and D metabolism. A signature of 91 enhancers distinguished between children with severe and mild asthma as well as controls. Conclusions: Gene regulatory enhancers were identified in leukocytes with potential roles related to severe and mild asthma in children. Enhancers hosting known SNPs give the opportunity to formulate mechanistic hypotheses about the functions of these SNPs.

Absolute counts of leukocyte subsets in peripheral blood in pregnancies complicated by preterm prelabour rupture of membranes.

BACKGROUND: This study aimed to assess variations in the absolute counts of various leukocyte subsets in the peripheral blood of women with pregnancies affected by preterm prelabour rupture of membranes (PPROM), in relation to the presence of intra-amniotic inflammation (IAI). METHODS: The study included fifty-two women with singleton pregnancies experiencing PPROM. Absolute counts of different leukocyte subpopulations, such as granulocytes, monocytes, lymphocytes, T cells and their subsets, B cells and their subsets, and NK cells and their subsets, were measured in maternal peripheral blood samples using multicolour flow cytometry. IAI was identified by elevated concentrations of interleukin 6 (IL-6) in the amniotic fluid, which was collected through transabdominal amniocentesis. RESULTS: Women with IAI exhibited higher absolute counts of leukocytes (p = 0.003), granulocytes (p = 0.008), and monocytes (p = 0.009). However, the presence of IAI did not significantly affect the absolute counts of lymphocytes or their subpopulations. CONCLUSIONS: The study found that IAI is associated with changes in the absolute counts of leukocytes from the innate immunity compartment in the peripheral blood of women with pregnancies complicated by PPROM. Conversely, it does not significantly alter the counts of cells from the adaptive immune system. The changes observed may reflect the natural, temporal, and localised characteristics of IAI.

Preterm birth is the most serious complication in contemporary perinatal medicine. Preterm birth, which is defined as a labour before the completion of 37 weeks of pregnancy, is often accompanied by premature rupture of the amniotic membranes and drainage of amniotic fluid. Such a situation is often complicated by inflammation, which adversely affects the health of the foetus. A number of procedures and markers have been developed for the diagnosis of inflammation, but they are determined from hard-to-reach amniotic fluid. It is therefore appropriate to try to find reliable markers of inflammation in the much more accessible maternal peripheral blood. Such a marker can be increased numbers of leukocytes, which have been repeatedly investigated in this context. However, little attention is directed to other leukocyte populations and especially to various lymphocyte subpopulations. This study aimed to test changes in absolute counts of different types of leukocytes and lymphocyte subpopulations in women with premature rupture of membranes with respect to ongoing inflammation. The results of the study showed that inflammation is accompanied by increased numbers of leukocytes, granulocytes and monocytes, however, the results did not show significant changes in the number of lymphocytes and their subpopulations.

Differential composition and yield of leukocytes isolated from various blood component leukoreduction filters.

In Flanders, an estimated 300,000 leukoreduction filters are discarded as biological waste in the blood establishment each year. These filters are a possible source of fresh donor leukocytes for downstream purposes including research. We investigated leukocyte isolation from two types of filters either used for the preparation of platelet concentrates (PC-LRF) or erythrocyte concentrates (EC-LRF). Outcome parameters were leukocyte yield, differential count, turnaround time and effect of storage conditions. Leukocytes were harvested by reverse flow of a buffer solution. Control was the gold standard density gradient centrifugation of buffy coats. Total leukocyte number isolated from PC-LRF (1049 (± 40) x 106) was almost double that of control (632 (± 66) x 106) but the differential count was comparable. Total leukocyte number isolated from EC-LRF (78 (± 9) x 106) was significantly lower than control, but the sample was specifically enriched in granulocytes (81 ± 4%) compared to control (30 ± 1%). Isolation of leukocytes from either PC- or EC-LRF takes 20 min compared to 240 min for control density gradient centrifugation. Leukocyte viability is optimal when harvested on day 1 post donation (95 ± 0.9%) compared to day 3 (76.4 ± 2.4%). In conclusion, our study demonstrates that leukoreduction filters from specific blood component processing are easy to use and present a valuable source for viable leukocytes of all types.

Stiffness regulates dendritic cell and macrophage subtype development and increased stiffness induces a tumor-associated macrophage phenotype in cancer co-cultures.

Mechanical properties of tissues including their stiffness change throughout our lives, during both healthy development but also during chronic diseases like cancer. How changes to stiffness, occurring during cancer progression, impact leukocytes is unknown. To address this, myeloid phenotypes resulting from mono- and cancer co-cultures of primary murine and human myeloid cells on 2D and 3D hydrogels with varying stiffnesses were analyzed. On soft hydrogels, conventional DCs (cDCs) developed, whereas on stiff hydrogels plasmacytoid DCs (pDCs) developed. Soft substrates promoted T cell proliferation and activation, while phagocytosis was increased on stiffer substrates. Cell populations expressing macrophage markers CD14, Ly6C, and CD16 also increased on stiff hydrogels. In cancer co-cultures, CD86+ populations decreased on higher stiffnesses across four different cancer types. High stiffness also led to increased vascular endothelial growth factor A (VEGFA), matrix metalloproteinases (MMP) and CD206 expression; 'M2' markers expressed by tumor-associated macrophages (TAMs). Indeed, the majority of CD11c+ cells expressed CD206 across human cancer models. Targeting the PI3K/Akt pathway led to a decrease in CD206+ cells in murine cultures only, while human CD86+ cells increased. Increased stiffness in cancer could, thus, lead to the dysregulation of infiltrating myeloid cells and shift their phenotypes towards a M2-like TAM phenotype, thereby actively enabling tumor progression. Additionally, stiffness-dependent intracellular signaling appears extremely cell context-dependent, potentially contributing to the high failure rate of clinical trials.

Effects of parity and early pregnancy on peripheral blood leukocytes in dairy cattle.

Subfertility remains a major problem in the dairy industry. Only 35-40% of high-yielding dairy cows and 55-65% of nonlactating heifers become pregnant after their first service. The immune system plays a critical role in the establishment of pregnancy. However, it can also create challenges for embryo survival and contribute to reduced fertility. We conducted 2 separate experiments to characterize changes in subsets of peripheral blood leukocytes (PBL) and their phenotype over the estrous cycle and early pregnancy in heifers and cows. We used flow cytometry and RT-qPCR to assess protein and mRNA expression of molecules important for immune function. We observed that CD14+ monocytes and CD3+ T cells tended to be affected by pregnancy status in heifers, whereas CD8B+ lymphocytes and NCR1+ natural killer (NK) cells were affected during early pregnancy in cows. Changes in expression of immune function proteins appeared to be greater in heifers than cows. To compare the most striking differences between heifers and cows observed in the initial experiments, we conducted a third experiment where PBL sampled from heifers and cows were simultaneously collected and analyzed under the same experimental conditions. Our results indicate that, compared with heifers, cows had greater mRNA expression of proinflammatory cytokines (IFNG and IL6) and AHR protein along with greater percentage of MM20A+ neutrophils and myeloid cells expressing SIRPA, ITGAM and ITGAX. Moreover, animals that failed to become pregnant showed altered expression of anti-inflammatory molecules compared with cyclic and pregnant animals. Overall, these findings support the hypothesis that early pregnancy signaling alters the phenotype of immune cells in the peripheral blood and that there are differences in the peripheral immune response to pregnancy between cows and heifers. Because cows have lower conception rates than heifers, it is possible that a more proinflammatory immune status in peripheral blood may play a role in embryo loss.

Neuroimmune circuits in the plaque and bone marrow regulate atherosclerosis.

Atherosclerosis remains the leading cause of death globally. Although its focal pathology is atheroma that develops in arterial walls, atherosclerosis is a systemic disease involving contributions by many organs and tissues. It is now established that the immune system causally contributes to all phases of atherosclerosis. Recent and emerging evidence positions the nervous system as a key modulator of inflammatory processes that underly atherosclerosis. This neuro-immune crosstalk, we are learning, is bidirectional, and immune regulated afferent signaling is becoming increasingly recognized in atherosclerosis. Here, we summarize data and concepts that link the immune and nervous systems in atherosclerosis by focusing on two important sites, the arterial vessel and the bone marrow.

Heterogeneous Patterns of Endothelial NF-κB p65 and MAPK c-Jun Activation, Adhesion Molecule Expression, and Leukocyte Recruitment in Lung Microvasculature of Mice with Sepsis.

BACKGROUND: Sepsis is an uncontrolled systemic inflammatory response to an infection that can result in acute failure of the function of the lung called acute respiratory distress syndrome. Leukocyte recruitment is an important hallmark of acute lung failure in patients with sepsis. Endothelial cells (EC) participate in this process by facilitating tethering, rolling, adhesion, and transmigration of leukocytes via adhesion molecules on their cell surface. In in vivo studies, endothelial nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mitogen-activated protein kinase (MAPK) c-Jun intracellular signal transduction pathways were reported to regulate the expression of adhesion molecules. METHODS: Mice underwent cecal ligation and puncture (CLP) to induce polymicrobial sepsis and were sacrificed at different time points up to 72 h after sepsis onset. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses were used to determine the kinetics of nuclear localization of p65 and c-Jun in EC, expression and location of adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Furthermore, the extent and location of leukocyte recruitment were assessed based on Ly6G staining of neutrophils, cluster determinant (CD) 3 staining of T lymphocytes, and CD68 staining of macrophages. RESULTS: In all pulmonary microvascular beds, we identified p65 and c-Jun nuclear accumulation in a subset of endothelial cells within the first 24 h after CLP-sepsis initiation. E-selectin protein was expressed in a subset of microvessels at 4 and 7 h after sepsis initiation, while VCAM-1 was expressed in a scattered pattern in alveolar tissue and microvessels, without discernible changes during sepsis development. CLP-induced sepsis predominantly promoted the accumulation of neutrophils and T lymphocytes 4 and 7 h after disease onset. Neutrophil accumulation occurred in all pulmonary microvascular beds, while T lymphocytes were present in alveolar tissue and postcapillary venules. Taken together, nuclear localization of p65 and c-Jun in EC and neutrophil recruitment could be associated with induced E-selectin expression in the pulmonary microvessels in CLP-septic mice at the early stage of the disease. In alveolar capillaries, on the other hand, activation of these molecular pathways and leukocyte accumulation occurred in the absence of E-selectin or VCAM-1. CONCLUSIONS: Endothelial activation and leukocyte recruitment in sepsis-induced lung injury are regulated by multiple, heterogeneously controlled mechanisms, which vary depending on the type of microvascular bed involved.

Ovarian leukocytes: Association with follicular persistence and cyst formation in dairy cows.

One of the initial causes of cystic ovarian disease (COD) is a failure in the normal ovulation mechanism. This study aimed to characterize the populations of immune cells (T-lymphocytes, B-lymphocytes, monocytes-macrophages and granulocytes) present in the ovary of cows with COD and induced follicular persistence, and evaluate their relation with follicular persistence and cyst formation. The follicular persistence model was developed using a progesterone (P4) slow-release intravaginal device, to obtain subluteal concentrations of P4. Results evidenced that T-lymphocytes, B-lymphocytes and monocytes-macrophages in the cortex, medulla, and theca externa and interna of dominant follicles were higher in the control group than in the COD and all persistence groups. Granulocytes in the medulla and theca externa of dominant follicles were lower in the control group than in the COD group, and those in the cortex and medulla were lower in the control group than in the persistence groups. The presence of T-lymphocytes, B-lymphocytes and granulocytes in the follicular fluid was abundant, especially that of granulocytes, without differences between control and COD cows. These results suggest that the immune system potentially plays a role in the local mechanisms of COD pathogenesis in dairy cows. In spontaneous COD and in our follicular persistence model, the distribution of the cells studied was different from that in the control group. However, to our knowledge, this is the first report describing the presence of immune cells in bovine follicular fluid samples and the expression of steroid hormone receptors in infiltrating immune cells in the bovine ovary.

Prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: communication from the SSC of the ISTH.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Andrographolide Inhibits Expression of NLPR3 Inflammasome in Canine Mononuclear Leukocytes.

Inflammasomes are multiprotein complexes that trigger processes through caspase-1 activation, leading to the maturation of proinflammatory cytokines, such as IL-1ß and IL-18. The gene encoding the inflammasome stimulatory protein NLRP3 is conserved in canines. Caspase-1/4 homologues have been identified in multiple carnivores, including canines, and caspase-1 activity has been shown in humans. The NLRP3 inflammasome has also been described in some canine inflammatory diseases. Andrographolide, a labdane diterpene, is the principal active ingredient in the herb Andrographis paniculate. The objective of this study was to determine the effect of andrographolide on the gene expression of the components of the NLRP3 inflammasome, proinflammatory cytokines, and IL-1ß secretion in canine peripheral blood mononuclear cells. For this, MTT assays and real-time PCR were employed to assess the cytotoxicity and gene expression. Further, an ELISA test was performed to measure the IL-1ß concentration. The findings reveal that andrographolide significantly reduces the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Additionally, it decreases the secretion of IL-1ß and other proinflammatory cytokines, including IL-6, IL-8, and TNF-α. The results show that andrographolide decreases the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Andrographolide also reduces proinflammatory cytokines expression, and decreases IL-1ß secretion. This indicates that andrographolide can interfere with the activation and function of the inflammasome, resulting in a decrease in the inflammatory response in canines. Research in this area is still budding, and more studies are necessary to fully understand andrographolide's mechanisms of action and its therapeutic potential in relation to the NLRP3 inflammasome in dogs.

Lebanese cannabis oil as a potential treatment for acute myeloid leukemia: In vitro and in vivo evaluations.

ETHNOPHARMACOLOGICAL RELEVANCE: The Cannabis sativa L. ssp. indica (Lam.) plant has been historically utilized as a natural herbal remedy for the treatment of several ailments. In Lebanon, cannabis extracts have long been traditionally used to treat arthritis, diabetes, and cancer. AIM OF THE STUDY: The current study aims to investigate the anti-cancer properties of Lebanese cannabis oil extract (COE) on acute myeloid leukemia using WEHI-3 cells, and a WEHI-3-induced leukemia mouse model. MATERIALS AND METHODS: WEHI-3 cells were treated with increasing concentrations of COE to determine the IC50 after 24, 48 and 72-h post treatment. Flow cytometry was utilized to identify the mode of cell death. Western blot assay was performed to assess apoptotic marker proteins. In vivo model was established by inoculating WEHI-3 cells in BALB/c mice, and treatment commencing 10 days post-inoculation and continued for a duration of 3 weeks. RESULTS: COE exhibited significant cytotoxicity with IC50 of 7.76, 3.82, and 3.34 µg/mL at 24, 48, and 72 h respectively post-treatment. COE treatment caused an induction of apoptosis through an inhibition of the MAPK/ERK pathway and triggering a caspase-dependent apoptosis via the extrinsic and intrinsic modes independent of ROS production. Animals treated with COE exhibited a significantly higher survival rate, reduction in spleen weight as well as white blood cells count. CONCLUSION: COE exhibited a potent anti-cancer activity against AML cells, both in vitro and in vivo. These findings emphasize the potential application of COE as a chemotherapeutic adjuvant in treatment of acute myeloid leukemia.